Genetic alterations are detected in all tumor cells. These alterations, occurring at the level of DNA, are transcribed and translated to generate altered proteins that in many instances drive cancer. These altered proteins can sometime contribute to immune recognition by T and B cells evoking activation of the immune response, which can lead to the elimination of tumor cells expressing the altered proteins [1-3].
Tumor cells, including malignant tumor cells or cancer cells, accumulate a large number of somatic mutations, from as low as ten, to as high as thousands depending on the cancer type. Only a subset of these mutations can evoke an immune response. Identifying such mutations can lead to the generation of therapeutic vaccines that can be given to patient as a polypeptide or as nucleic acids (both DNA and RNA) [4].
For a mutation to be recognized as foreign, the mutant amino acid should be present as part of a peptide that binds class I or class II major histocompatibility complex (MHC or alternatively known as human leukocyte antigen or HLA in human) molecules and be presented on the surface of antigen presenting cells (professional APCs). The MHC- or HLA-bound peptide interacts with the T-cell receptor (TCR) expressed on the surface of T cells. Productive binding with the TCR activates T-cells, which can kill tumor cells directly through its cytolytic activity (CD8+ cytotoxic T-cells) or perform helper function (CD4+ helper T-cells) to induce antibody production. In this context, the definition of an immunogenic peptide is restricted to peptides that can interact with CD8+ or CD4+ T cells. For the interaction to happen, the peptide must be presented on the surface of cells in complex with MHC or HLA class I or class II proteins. The MHC class I- or HLA class I-bound peptide interacts with CD8+ T cells, and the MHC class II- or HLA class II-bound peptide interacts with CD4+ T cells. Although MHC or HLA binding and surface presentation is required for T cell activation, but, the displayed peptide bound to MHC or HLA proteins on the surface of cell is necessary but not sufficient for T cell activation as TCR must also interact with the displayed peptide. Most peptides presented on the cell surface in complex with MHC or HLA fail to engage T cells and therefore are not immunogenic [5]. Immunogenicity require not only peptide-binding and display by MHC class I or class II proteins but also binding of the MHC class I or class II-displayed peptide by TCR of the CD8+ T-cell or CD4+ T-cell, respectively [6]. While much is known about the rules governing peptide binding by MHC or HLA molecules, little is known about the rules governing peptide binding by TCR, other than that the rules governing peptide binding by TCR are different from peptide binding by MHC or HLA proteins.
Class I HLA proteins are encoded by HLA-A, HLA-B and HLA-C genes. These proteins bind peptides of 8-11 amino acids in length, with the preferred length being 9 amino acids long. The peptide binding groove of class HLA is formed by two alpha helices supported by an anti-parallel beta sheet. The peptide-binding groove is deeper compared to class II HLA molecules and requires residues to be projected outside the binding groove to make interactions with the TCR [7].
Peptides bind to class HLA molecules in a multistep process. The steps are as follows: 1) generation of protein fragments by immunoproteasomal or proteasomal processing as part of the natural turnover of proteins in cells [8]; 2) Entry of the protein fragment into the lumen of the endoplasmic reticulum by binding to peptide transporters (TAP) [9]; 3) Binding to the peptide-binding groove of the class I HLA molecules; 4) Transport through vesicles to the cell surface and 5) presentation on the surface of cells [10] [11].
In the case of endogenous proteins, such as altered proteins in tumor or cancer cells, these proteins being produced intracellularly by the cell do not require cellular uptake. As such, peptides derived by immunoproteasomal or proteasomal processing as part of the natural turnover of proteins in cells may be displayed by class I MHC or HLA molecules in all cell types in which the altered protein is expressed by the cell. In contrast, in the case of a peptide used in tumor or cancer vaccine, the peptide is exogenous to the cell and must be taken up by professional antigen-presenting cells in a process called cross-presentation in order to be displayed by class I MHC or HLA proteins [12-14]. The peptide used in tumor or cancer vaccine is longer than the peptide displayed by class I MHC or HLA proteins, as the peptide is taken up by the cell and undergo proteolysis to produce shorter peptide(s). Equal number of amino acids are added to the amino- and carboxy-termini, so as to extend the length of the final peptide displayed by class I MHC or HLA proteins. Typically, five to eighteen amino acids are added to each end of the 8-11 amino acid long peptide displayed on cell surface by class I MHC or HLA proteins, such that the peptide formulated in the tumor or cancer vaccine is approximately 18 to 47 amino acids in length. The upper limit of peptide length in tumor or cancer vaccine is less than or equal to 50 amino acids. The antigen-presenting cells capable of cross presentation are professional antigen-presenting cells and include dendritic cells (primarily), macrophages, and B lymphocytes.
The binding of MHC-peptide complex to the CD8+ T cells, henceforth referred to as cytolytic or cytotoxic T cells (CTLs) activates a series of signaling pathways in CTLs resulting in their expansion to generate a population of effector CTLs. These CTLs will recognize tumor cells displaying the mutant peptide on their surface and kill them by apoptosis. Therefore, peptides derived from cancer mutations that are capable of mounting a CTL response can be used as cancer vaccines for treating cancer patients [15].
Two studies have demonstrated that immunogenic peptides can provide long term benefit to cancer patients when used as monotherapy [16, 17]. Therefore, accurate identification of immunogenic peptides from tumor-derived mutant protein can provide an avenue of treatment for cancer patients [18] [19]. However, the lack of efficient method for identifying bonafide immunogenic peptides have not only increased the cost of vaccination, but also increased the uncertainty of whether the vaccine will deliver the desired effect of inducing an anti-tumor response.
Next generation sequencing technology can catalogue all tumor mutations from a patient's tumor cells rapidly. However, identifying immunogenic peptides derived from such mutations is still a formidable challenge. The challenge comes from the fact that accurate methods of selecting immunogenic peptides from a pool of immunogenic and non-immunogenic peptides [20] [18].
Most screening platform uses HLA-binding prediction as a measure of immunogenicity [21]. The prediction can be further confirmed by actual detection of the peptide on the cell surface by mass spectrometry [5]. However, surface presentation of a peptide in complex with HLA is not an indication of immunogenicity. For a peptide to be immunogenic, the peptide presented on the surface of cells must engage T cell receptor. There is a need in the art for a high throughput methodology for prediction of immunogenic peptide for cancer therapy.